Study On The Recombinant Human Basic/Acidic Fibroblast Growth Factor Expressed In Rice Endosperm

Molecular pharming is to use plant or animal cells as bioreactors to produce theraputical proteins. The critical technology of molecular pharming is to large-scalely and cost-effectively produce recombination theraputical proteins through transgenic approaches. In this reaserch, we used the expression platform that well established in our laboratory and successfully expressed recombinant human basic/acidic fibroblast growth factors. The major results were obtained as following:1^We used the rice endosperm expression platform well established in our Laboratory to express a recombinant human basic fibroblst growth factor (rhbFGF). A hbFGF gene was synthesized using rice-prefferred condons and inserted the expression vector containing Gtl3a promoter and signal peptide sequence. Then the expression vector was co-transfromed with the pOsPMP05through Agrobacterium-mediated transformation using a rice variety TP309. Eight homozygous lines were obtained by analysis of Western blot analysis. A transgenic line277-179with the highest expression level of OsrbFGF (Oryza sativa recombinant basic fibroblast growth factor) was obtained, expression level reach up to185.66mg/kg, and the soluble OsrbFGF is about9.55%of total OsrbFGF.2、The N-terminal sequence analysis of OsrbFGF showed that one amino acid, Ala, was missed, which could be cleaved by endogenous peptidase. The results of molecular mass and isoelectric point indicated that OsrbFGF, compared to native bFGF (molecular mass-17kDa, isoelectric point9.6), has differences in two molecular masses of16kDa and17kDa and two isoelectric points (9.55and8.93)3、The genetic analysis indicated that two transgenic lines,277-122and277-179, have two insertion sites, and277-223and277-238have a single insertion site. Three loci in transgenic line of277-122, two loci in277-179and one locus in277-223and277-238 were determined by Southern blotting analysis.4、Further transparent electro-miscroscopic observation indicated that morphology change of protein bodies was observed in the transgenic lines. Immuno-TEM showed that OsrbFGF localized in protein body I, protein body II and apoplast, indicating OsrbFGF undergo a default trafficking pathway.5、We explored the purification protocol to isolate and purify OsrbFGF from transgenic seeds. A single purification step protocol was established. The purity of OsrbFGF showed a single band in SDS-PAGE. The resulting purity of OsrbFGF reached up to>95%. Endotoxin contents is<1EU/μg by a step of ultrafiltration using5kDa or100kDa filters. The resulting yield of OsrbFGF was8.33mg/kg of brown rice with a recovery of4.49%.6^We further explored the biological actiovity of OsrbFGF using NIH/3T3Cell proliferation assay. The results showed that the stimulating NIH/3T3Cell proliferation activitiy of OsrbFGF reached1.04×106IU/mg as a dose-dependent fashion, showing the same stimulation of cell proliferation as commercialized rbFGF.7、The wound healing activity in vivo indicated that OsrbFGF could accelerate wound healing by stimulating cell proliferation and improving the expression of CD68in the early stage of treatment. It then decreased the expression to prevent the generation of scars and cell proliferation when the wound closed at the late stage of the treatment.8、We expressed a recombinant human acidic fibroblast growth factor (rhaFGF)using the same strategy in transgenic rice endosperm. A haFGF gene was synthesized using rice-prefferred condons and inserted the expression vector containing GtJ3a promoter and signal peptide sequence. Total of53independent transgenic plants were obtained and confirmed by PCR amplification. Twelve transgenic lines were determined to express OsraFGF (Oryza saliva recombinant acidic fibroblast growth factor) protein by Western blot analysis.9、Genetic analysis showed that genetic segregation of107-33-2-3in T1seeds fits to15:1Mendel segregation, indicating two loci insertion sites in this line. Southern blot result displayed that three copies of haFGF gene inserted into rice genome.10、The TEM observation showed that the morphology of the protein bodies in the ecdosperm do not changed.11、We preliminarily explored a protocol for extraction and purififcation of OsraFGF from transgenic rice seeds.